Astaxanthin is reportedly distributed in a wide variety of organisms such as animals (e.g., birds, such as flamingo and scarlet ibis; fish, such as rainbow trout and salmon), algae and microorganisms. It is also reported that astaxanthin has a strong antioxidation property against oxygen radicals, which is believed to be pharmaceutically useful for protecting living cells against some diseases such as a cancer. Moreover, from a commercial prospective, there is an increasing demand for astaxanthin as a coloring reagent especially in the fish farming industry, such as salmon farming, because astaxanthin imparts a distinctive orange-red coloration to the fish and contributes to consumer appeal.
Phaffia rhodozyma is known as a carotenogenic yeast strain which produces astaxanthin specifically. Different from the other carotenogenic yeast, Rhodotorula species, such as Phaffia rhodozyma (P. rhodozyma) can ferment some sugars such as D-glucose. This is a commercially important feature. In a recent taxonomic study, the sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated as Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast: 11, 101-110, 1995). Some strain improvement studies to obtain hyper-producers of astaxanthin from P. rhodozyma have been conducted, but such efforts have been restricted to conventional methods including mutagenesis and protoplast fusion in this decade.
Recently, Wery et al. reportedly developed a host vector system using P. rhodozyma in which a non-replicable plasmid was integrated into the genome of P. rhodozyma at the locus of a ribosomal DNA in multiple copies (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported vectors for obtaining a transformant of P. rhodozyma, as well as its three carotenogenic genes which code for the enzymes that catalyze the reactions from geranylgeranyl pyrophosphate to β-carotene (International patent WO97/23633).
It has been reported that the carotenogenic pathway from a general metabolite, acetyl-CoA consists of multiple enzymatic steps in carotenogenic eukaryotes as shown in FIG. 1. In this pathway, two molecules of acetyl-CoA are condensed to yield acetoacetyl-CoA which is converted to 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) by the action of 3-hydroxymethyl-3-glutaryl-CoA synthase. Next, 3-hydroxy-3-methylglutaryl-CoA reductase converts HMG-CoA to mevalonate, to which two molecules of phosphate residues are then added by the action of two kinases (mevalonate kinase and phosphomevalonate kinase). Mevalonate pyrophosphate is then decarboxylated by the action of mevalonate pyrophosphate decarboxylase to yield isopentenyl pyrophosphate (IPP) which becomes a building unit for a wide variety of isoprene molecules which are necessary in living organisms. This pathway is designated the “mevalonate pathway” taken from its important intermediate, mevalonate.
In this pathway, IPP is isomerized to dimethylaryl pyrophosphate (DMAPP) by the action of IPP isomerase. Then, IPP and DMAPP are converted to a C10 unit, geranyl pyrophosphate (GPP) by a head to tail condensation. In a similar condensation reaction between GPP and IPP, GPP is converted to a C15 unit, farnesyl pyrophosphate (FPP) which is an important substrate of cholesterol in animals, of ergosterol in yeast, and of the farnesylation of regulation proteins, such as the RAS protein. In general, the biosynthesis of GPP and FPP from IPP and DMAPP are catalyzed by one enzyme called FPP synthase (Laskovics et al., Biochemistry, 20, 1893-1901, 1981).
On the other hand, in prokaryotes such as eubacteria, isopentenyl pyrophosphate is reportedly synthesized in a different pathway via 1-deoxyxylulose-5-phosphate from pyruvate which is absent in yeast and animals (Rohmer et al., Biochem. J., 295, 517-524, 1993).